This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Hypothemycin is a natural product known to irreversibly inhibit a subset of kinases that contain a non-conserved cysteine in their active site. Although its in vitro selectivity profile is known, what it targets in a more "realistic", in vivo context remains mostly unknown. We have recently synthesized an analog of hypothemycin that mirrors the activity of its parent and allows us to use Click Chemistry to identify those targets that it covalently labels. In collaboration with the UCSF Mass Spectrometry Facility, we have used this molecule to identify several essential kinases in Trypanosoma brucei, the causative agent of African sleeping sickness, of which only one was known and studied before. We continue to use this molecule to better understand the roles of these targets in the parasite. Also by applying the methods developed through our work with T. brucei, we plan to develop a list of human kinases labeled by our hypothemycin probe in vivo and compare that to what has been generated in vitro. This allows us to explore the in vivo selectivity profiles of other small molecule kinase inhibitors, including approved drugs, and begin to resolve the disconnect between in vitro and in vivo profiles. Mass spectrometry provides the sensitivity necessary to detect low abundance kinases and the ability to identify multiple kinases within the same sample and to quantify our results. This makes mass spectrometry an important tool for us to effectively explore and understand how kinase inhibitor selectivity in vivo compares to in vitro systems.